U.S. patent application number 17/172553 was filed with the patent office on 2021-08-12 for nanopore device and methods of detecting and classifying charged particles using same.
The applicant listed for this patent is PALOGEN, INC.. Invention is credited to Kyung Joon Han, Bita Karimirad.
Application Number | 20210247378 17/172553 |
Document ID | / |
Family ID | 1000005481362 |
Filed Date | 2021-08-12 |
United States Patent
Application |
20210247378 |
Kind Code |
A1 |
Karimirad; Bita ; et
al. |
August 12, 2021 |
NANOPORE DEVICE AND METHODS OF DETECTING AND CLASSIFYING CHARGED
PARTICLES USING SAME
Abstract
A method of determining an oligonucleotide methylation
percentage includes providing a 3D nanopore device having top and
bottom chambers, and a 3D nanochannel array disposed therein. The
method also includes purifying an oligonucleotide, and
functionalizing the 3D nanochannel array by coupling an
oligonucleotide probe. The method further includes forming an
oligonucleotide solution having a known concentration, and adding
the oligonucleotide solution to the top and bottom chambers.
Moreover, the method includes placing top and bottom electrodes in
the top and bottom chambers respectively, applying an
electrophoretic bias between the top and bottom electrodes,
applying a selection bias across first and second gating
nanoelectrodes, applying a sensing bias through a sensing
nanoelectrode in the 3D nanopore device. In addition, the method
includes detecting an output current from the sensing
nanoelectrode, and analyzing the output current from the sensing
nanoelectrode to determine a methylation percentage of the
oligonucleotide.
Inventors: |
Karimirad; Bita; (Seoul,
KR) ; Han; Kyung Joon; (Palo Alto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PALOGEN, INC. |
Palo Alto |
CA |
US |
|
|
Family ID: |
1000005481362 |
Appl. No.: |
17/172553 |
Filed: |
February 10, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62972415 |
Feb 10, 2020 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/48721 20130101;
C12Q 1/6827 20130101 |
International
Class: |
G01N 33/487 20060101
G01N033/487; C12Q 1/6827 20060101 C12Q001/6827 |
Claims
1. A method of determining an oligonucleotide methylation
percentage, comprising: providing a 3D nanopore device having top
and bottom chambers, and a 3D nanochannel array disposed in the top
and bottom chambers such that the top and bottom chambers are
fluidly coupled by a plurality of nanochannels in the 3D
nanochannel array; purifying an oligonucleotide; functionalizing
the 3D nanochannel array by coupling an oligonucleotide probe to an
inner surface of the 3D nanopore device defining the nanochannel,
wherein the oligonucleotide probe is complementary to the
oligonucleotide; adding the purified oligonucleotide to DI water to
form an oligonucleotide solution having a known concentration;
adding the oligonucleotide solution including the oligonucleotide
to the top and bottom chambers; placing top and bottom electrodes
in the top and bottom chambers respectively; applying an
electrophoretic bias between the top and bottom electrodes;
applying a selection bias across first and second gating
nanoelectrodes in the 3D nanopore device to direct flow of the
oligonucleotide through a nanochannel of the plurality of
nanochannels; applying a sensing bias through a sensing
nanoelectrode in the 3D nanopore device; detecting an output
current from the sensing nanoelectrode; and analyzing the output
current from the sensing nanoelectrode to determine a methylation
percentage of the oligonucleotide.
2. The method of claim 1, further comprising functionalizing the 3D
nanochannel array by coupling a second oligonucleotide probe to an
inner surface of the 3D nanopore device defining a second
nanochannel, wherein the second oligonucleotide probe is different
from the oligonucleotide probe.
3. The method of claim 1, wherein analyzing the output current from
the sensing electrode to determine a methylation percentage of the
oligonucleotide comprises comparing the output current and the
sensing bias to corresponding values in a reference table for the
known concentration.
4. The method of claim 1, wherein analyzing the output current from
the sensing electrode to determine a methylation percentage of the
oligonucleotide comprises using an effect of methylation on a
charge of a phosphate backbone of the oligonucleotide.
5. The method of claim 1, further comprising: applying a second
sensing bias through the sensing nanoelectrode in the 3D nanopore
device; detecting a second output current from the sensing
nanoelectrode; analyzing the second output current from the sensing
nanoelectrode to determine a second methylation percentage of the
oligonucleotide; and comparing the second methylation percentage of
the oligonucleotide to the methylation percentage of the
oligonucleotide to confirm the methylation percentage of the
oligonucleotide.
6. The method of claim 1, wherein the oligonucleotide is an RNA
molecule fragment or a DNA molecule fragment.
7. (canceled)
8. The method of claim 1, wherein the oligonucleotide is extracted
from cell free DNA, tissue or cell culture medium, serum, urine,
plasma, or saliva.
9.-10. (canceled)
11. The method of claim 1, wherein charge carriers in the 3D
nanopore device comprise the DI water, H+ ions, and OH- ions.
12. The method of claim 1, further comprising: removing the
oligonucleotide solution including the oligonucleotide from the top
and bottom chambers; purifying a second oligonucleotide;
functionalizing the 3D nanochannel array by coupling a second
oligonucleotide probe to an inner surface of the 3D nanopore device
defining the nanochannel, wherein the second oligonucleotide probe
is complementary to the second oligonucleotide; adding the purified
second oligonucleotide to DI water to form a second oligonucleotide
solution having a known concentration; adding the second
oligonucleotide solution including the second oligonucleotide to
the top and bottom chambers; applying the electrophoretic bias
between the top and bottom electrodes; applying the selection bias
across the first and second gating nanoelectrodes in the 3D
nanopore device to direct flow of the second oligonucleotide
through the nanochannel; applying the sensing bias through the
sensing nanoelectrode in the 3D nanopore device; detecting a second
output current from the sensing nanoelectrode; and analyzing the
second output current from the sensing nanoelectrode to determine a
methylation percentage of the second oligonucleotide.
13. The method of claim 1, further comprising: applying a second
selection bias across third and fourth gating nanoelectrodes in the
3D nanopore device to direct flow of a second oligonucleotide
through a second nanochannel of the plurality of nanochannels;
applying a second sensing bias through a second sensing
nanoelectrode in the 3D nanopore device; detecting a second output
current from the second sensing nanoelectrode; and analyzing the
second output current from the second sensing nanoelectrode to
determine a methylation percentage of the second
oligonucleotide.
14. The method of claim 1, wherein analyzing the output current
from the sensing electrode to determine a methylation percentage of
the oligonucleotide comprises differentiating between methyl
cytosine methylation and hydroxy methyl cytosine methylation.
15. The method of claim 1, further comprising comparing the
methylation percentage of the oligonucleotide to a library of
methylation patterns corresponding to known mutations to diagnose a
disease, wherein the disease is cancer, atherosclerosis, or
aging.
16. (canceled)
17. The method of claim 1, wherein the oligonucleotide probe is a
DNA probe, an RNA probe, or a protein probe.
18. The method of claim 1, further comprising analyzing the output
current from the sensing nanoelectrode to quantify a number of
methylation sites in the oligonucleotide.
19. The method of claim 1, further comprising applying a rate
control bias to a rate control nanoelectrode in the 3D nanopore
device to modulate a translocation rate of the oligonucleotide
through the nanochannel.
20. The method of claim 1, wherein the current is an electrode
current.
21. The method of claim 1, wherein the current is a tunneling
current.
22. The method of claim 1, wherein the first gating nanoelectrode
addresses a first end of the nanochannel, wherein the second gating
nanoelectrode addresses a second end of the nanochannel opposite
the first end, and wherein a sensing nanoelectrode addresses a
first location in the nanochannel between the first and second
ends.
23. The method of claim 1, further comprising alternatively
reversing the electrophoretic bias and the selection bias to direct
alternating flow of the oligonucleotides through the nanochannel
between the first and second gating nanoelectrodes.
24. The method of claim 1, wherein the 3D nanopore device is
integrated into a mobile application, a laptop computer, or a
desktop computer.
25. The method of claim 1, wherein the 3D nanopore device is
integrated into microfluidic device, a nanofluidic device, a
nanodevice, or a lab-on-chip system.
26. The method of claim 1, wherein the 3D nanopore device is
integrated into an all-in-one ASIC platform system for extraction
and sensing of the oligonucleotide.
27. The method of claim 1, further comprising: the 3D nanopore
device detecting hybridization of the oligonucleotide to the
oligonucleotide probe at a minimum concentration of the
oligonucleotide of about 10 femtomolar (limit of detection); and
the 3D nanopore device detecting hybridization of the
oligonucleotide to the oligonucleotide probe without amplification
of the oligonucleotide or use of PCR, wherein the 3D nanopore
device is integrated into a liquid biopsy panel platform to perform
detection without amplification of the oligonucleotide or use of
PCR.
28.-29. (canceled)
30. The method of claim 1, further comprising analyzing the output
current from the sensing nanoelectrode to determine a conformation
change of the oligonucleotide.
31. The method of claim 1, further comprising analyzing the output
current from the sensing nanoelectrode to determine a hydration
change of the oligonucleotide.
32.-33. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional
Application No. 62/972,415, filed on Feb. 10, 2020 under attorney
docket number PAL.30009.00 and, entitled "NANOPORE DEVICE AND
METHODS OF DETECTING AND CLASSIFYING CHARGED PARTICLES USING SAME,"
the contents of which are hereby expressly and fully incorporated
by reference in their entirety, as though set forth in full. This
application includes subject matter similar to the subject matter
described in co-owned U.S. Provisional Patent Application Ser. No.
62/566,313, filed on Sep. 29, 2017 under attorney docket number
165-101USIP and entitled "MANUFACTURE OF THREE DIMENSIONAL NANOPORE
DEVICE"; U.S. Provisional Patent Application Ser. No. 62/593,840,
filed on Dec. 1, 2017 under attorney docket number BTL.30002.00 and
entitled "NANOPORE DEVICE AND METHOD OF MANUFACTURING SAME"; U.S.
Provisional Patent Application Serial Number U.S. Provisional
Patent Application Ser. No. 62/612,534, filed on Dec. 31, 2017
under attorney docket number BTL.30003.00 and entitled "NANOPORE
DEVICE AND METHODS OF ELECTRICAL ARRAY ADDRESSING AND SENSING";
U.S. Provisional Patent Application Ser. No. 62/628,214, filed on
Feb. 8, 2018 under attorney docket number BTL.30004.00 and entitled
"BIOMEMORY FOR NANOPORE DEVICE AND METHODS OF MANUFACTURING SAME";
U.S. Utility patent application Ser. No. 16/147,362, filed on Sep.
26, 2018 under attorney docket number BTL.20001.00 and entitled
"NANOPORE DEVICE AND METHOD OF MANUFACTURING SAME"; U.S. Utility
patent application Ser. No. 16/237,570, filed on Dec. 31, 2018
under attorney docket number BTL.20003.00 and entitled "NANOPORE
DEVICE AND METHODS OF ELECTRICAL ARRAY ADDRESSING AND SENSING";
U.S. Provisional Patent Application Ser. No. 62/802,459, filed on
Feb. 7, 2019 under attorney docket number BTL.30004.01 and entitled
"BIOMEMORY FOR NANOPORE DEVICE AND METHODS OF MANUFACTURING SAME";
U.S. Provisional Patent Application Ser. No. 62/826,897, filed on
Mar. 29, 2019 under attorney docket number BTL.30006.00 and
entitled "NANOPORE DEVICE AND METHODS OF BIOSYNTHESIS USING SAME";
U.S. Utility patent application Ser. No. 16/524,033, filed on Jul.
27, 2019 under attorney docket number PAL.20005.00 and entitled
"NANOPORE DEVICE AND METHODS OF DETECTING CHARGED PARTICLES USING
SAME"; and U.S. Provisional Patent Application Ser. No. 62/874,766,
filed on Jul. 16, 2019 under attorney docket number PAL.30007.00
and entitled "NANOPORE DEVICE AND METHODS OF DETECTING AND
CLASSIFYING CHARGED PARTICLES USING SAME." The contents of the
above-mentioned applications are fully incorporated herein by
reference as though set forth in full.
FIELD OF THE INVENTION
[0002] The present invention relates generally to systems and
devices for characterizing epigenetic alterations, and methods of
detecting methylation patterns in genomes using such systems and
devices. In particular, the present invention relates to nanopore
sensors for detecting methylation patterns. The disclosed nanopore
sensors facilitate characterization of epigenetic alterations by
characterizing methylation patterns in genome-derived
oligonucleotides (e.g., detecting DNA methylation in genome-derived
oligonucleotides).
BACKGROUND
[0003] Early cancer detection and treatment can save millions of
lives. Accordingly, there is a need for a device (e.g., a
bedside/point of care detection system) and method for affordable,
rapid, accurate, and early detection of epigenetic alterations in
specific genes in a genome.
[0004] The etiology of cancer includes many types of genetic
changes that can lead to various alterations in cell functions. In
addition to genetic mutations, the etiology of cancer includes
epigenetic changes, which is directly related to the gene
expression and cancer. Detecting epigenetic changes can provide
effective screening techniques for cancer detection, and subsequent
treatment and cure by therapeutic intervention that conforms to the
particular early detected cancer.
[0005] Cytosine poly guanine island ("CpG island") methylation,
histone modifications, and reorganization of chromatin various
epigenetic mechanisms that regulate the activation and silencing of
genes. DNA methylation is an epigenetic mechanism that can control
DNA transcription and replication. Methylation patterns during
tissue specific cell type differentiation from stem cells, are
conserved during subsequent cell division to maintain the specific
cell type in newly formed tissue.
[0006] Many genes can be activated or silenced resulting in
carcinogenesis. While some mutations result in gene silencing, a
significant extent of carcinogenic gene silencing is the result of
alterations in DNA methylation. DNA methylation alteration in
multiple CpG sites in a CpG island, especially in protein promotor
regions, can lead to cancer via silencing of cancer reducing genes
(e.g., error correction enzymes).
[0007] Even the silencing of the genes caused by other processes
can be stabilized when the gene silencing is followed by promotor
methylation in the CpG islands. Methylation is very effective in
gene silencing. For instance, hypermethylation of a CpG island in a
promotor region is 10 times more effective in gene silencing
compared to a DNA mutation in the promotor region itself.
[0008] Accordingly, measurement of the methylation content of
target genes/sequences of interest can facilitate detection for
hypermethylation of specific sequences and diagnosis of related
disease, determination of disease prognosis, and/or monitoring of
disease. If the measurement of methylation can be completed in
around 10 minutes, such rapid measurement can facilitate point of
care diagnosis, prognosis determination, and disease monitoring.
Such measurement of methylation can facilitate other disease
monitoring (e.g., in addition to cancer), as long as the disease is
correlated with epigenetic alterations like DNA methylation.
[0009] Early experimental systems for nanopore based DNA sequencing
detected electrical behavior of ssDNA passing through an
.alpha.-hemolysin (.alpha.HL) protein nanopore. Since then,
nanopore based nucleic acid sequencing technology has been
improved. For instance, solid-state nanopore based nucleic acid
sequencing replaces biological/protein based nanopores with
solid-state (e.g., semiconductor, metallic gates) nanopores, as
described herein.
[0010] A nanopore is a small hole (e.g., with a diameter of in the
nanometer range that can detect the flow of charged particles
(e.g., methylated oligonucleotides, etc.) through the hole by the
change in the ionic current and/or tunneling current. Nanopore
technology is based on electrical sensing, which is capable of
detecting methylation of oligonucleotides in concentrations and
volumes much smaller than that required for other conventional
detection methods. Advantages of nanopore based methylated
oligonucleotide detection include long read length, plug and play
capability, and scalability. With advancements in semiconductor
manufacturing technologies, solid-state nanopores have become an
inexpensive and superior alternative to biological nanopores partly
due to the superior mechanical, chemical and thermal
characteristics, and compatibility with semiconductor technology
allowing the integration with other sensing circuitry and
nanodevices.
[0011] FIG. 1 schematically depicts a state-of-art solid-state
based 2-dimensional ("2D") nanopore sensing device 100. While, the
device 100 is referred to as "two dimensional," the device 100 has
some thickness along the Z axis. In order to address the some of
these drawbacks (sensitivity and some of the manufacturing cost) of
current state-of-art nanopore technologies, multi-channel nanopore
arrays which allows parallel processing of biomolecules may be used
to achieve amplification-free and rapid DNA methylation detection.
Examples of such multi-channel nanopore arrays are described in
U.S. Provisional Patent Application Ser. Nos. 62/566,313 and
62/593,840 and U.S. Utility patent application Ser. No. 16/524,033,
the contents of which have been previously incorporated by
reference.
[0012] As described herein, there is a need for a device (e.g., a
bedside/point of care detection system) and method for affordable,
rapid, accurate, and early detection of epigenetic alterations in
specific genes in a genome. In particular, there is a need for such
a device and method for detecting methylation of genomic DNA.
SUMMARY
[0013] Embodiments described herein are directed to nanopore based
electrically assisted methylation detection systems and methods of
detecting DNA methylation using same. In particular, the
embodiments are directed to various types (2D or 3D) of nanopore
based methylation detection systems, methods of using nanopore
array devices, and methods of methylation detection using same.
[0014] In one embodiment, a method of determining an
oligonucleotide methylation percentage includes providing a 3D
nanopore device having top and bottom chambers, and a 3D
nanochannel array disposed in the top and bottom chambers such that
the top and bottom chambers are fluidly coupled by a plurality of
nanochannels in the 3D nanochannel array. The method also includes
purifying an oligonucleotide. The method further includes
functionalizing the 3D nanochannel array by coupling an
oligonucleotide probe to an inner surface of the 3D nanopore device
defining the nanochannel, where the oligonucleotide probe is
complementary to the oligonucleotide. Moreover, the method includes
adding the purified oligonucleotide to DI water to form an
oligonucleotide solution having a known concentration. In addition,
the method includes adding the oligonucleotide solution including
the oligonucleotide to the top and bottom chambers. The method also
includes placing top and bottom electrodes in the top and bottom
chambers respectively. The method further includes applying an
electrophoretic bias between the top and bottom electrodes.
Moreover, the method includes applying a selection bias across
first and second gating nanoelectrodes in the 3D nanopore device to
direct flow of the oligonucleotide through a nanochannel of the
plurality of nanochannels. In addition, the method includes
applying a sensing bias through a sensing nanoelectrode in the 3D
nanopore device. The method also includes detecting an output
current from the sensing nanoelectrode. The method further includes
analyzing the output current from the sensing nanoelectrode to
determine a methylation percentage of the oligonucleotide.
[0015] In one or more embodiments, the method also includes
functionalizing the 3D nanochannel array by coupling a second
oligonucleotide probe to an inner surface of the 3D nanopore device
defining a second nanochannel, where the second oligonucleotide
probe is different from the oligonucleotide probe. Analyzing the
output current from the sensing electrode to determine a
methylation percentage of the oligonucleotide may include comparing
the output current and the sensing bias to corresponding values in
a reference table for the known concentration. Analyzing the output
current from the sensing electrode to determine a methylation
percentage of the oligonucleotide may include using an effect of
methylation on a charge of a phosphate backbone of the
oligonucleotide.
[0016] In one or more embodiments, the method also includes
applying a second sensing bias through the sensing nanoelectrode in
the 3D nanopore device. The method further includes detecting a
second output current from the sensing nanoelectrode. Moreover, the
method includes analyzing the second output current from the
sensing nanoelectrode to determine a second methylation percentage
of the oligonucleotide. In addition, the method includes comparing
the second methylation percentage of the oligonucleotide to the
methylation percentage of the oligonucleotide to confirm the
methylation percentage of the oligonucleotide.
[0017] In one or more embodiments, the oligonucleotide is an RNA
molecule fragment or a DNA molecule fragment. The oligonucleotide
may be extracted from cell free DNA, tissue, cell culture medium,
serum, urine, plasma, or saliva. Charge carriers in the 3D nanopore
device may include the DI water, H+ ions, and OH- ions.
[0018] In one or more embodiments, the method also includes
removing the oligonucleotide solution including the oligonucleotide
from the top and bottom chambers. The method further includes
purifying a second oligonucleotide. Moreover, the method includes
functionalizing the 3D nanochannel array by coupling a second
oligonucleotide probe to an inner surface of the 3D nanopore device
defining the nanochannel, where the second oligonucleotide probe is
complementary to the second oligonucleotide. In addition, the
method includes adding the purified second oligonucleotide to DI
water to form a second oligonucleotide solution having a known
concentration. The method also includes adding the second
oligonucleotide solution including the second oligonucleotide to
the top and bottom chambers. The method further includes applying
the electrophoretic bias between the top and bottom electrodes.
Moreover, the method includes applying the selection bias across
the first and second gating nanoelectrodes in the 3D nanopore
device to direct flow of the second oligonucleotide through the
nanochannel. In addition, the method includes applying the sensing
bias through the sensing nanoelectrode in the 3D nanopore device.
The method also includes detecting a second output current from the
sensing nanoelectrode. The method further includes analyzing the
second output current from the sensing nanoelectrode to determine a
methylation percentage of the second oligonucleotide.
[0019] In one or more embodiments, the method also includes
applying a second selection bias across third and fourth gating
nanoelectrodes in the 3D nanopore device to direct flow of a second
oligonucleotide through a second nanochannel of the plurality of
nanochannels. The method further includes applying a second sensing
bias through a second sensing nanoelectrode in the 3D nanopore
device. Moreover, the method includes detecting a second output
current from the second sensing nanoelectrode. In addition, the
method includes analyzing the second output current from the second
sensing nanoelectrode to determine a methylation percentage of the
second oligonucleotide.
[0020] In one or more embodiments, analyzing the output current
from the sensing electrode to determine a methylation percentage of
the oligonucleotide includes differentiating between methyl
cytosine methylation and hydroxy methyl cytosine methylation. The
method may also include comparing the methylation percentage of the
oligonucleotide to a library of methylation patterns corresponding
to known mutations to diagnose a disease. The disease may be
cancer, atherosclerosis, or aging.
[0021] In one or more embodiments, the oligonucleotide probe is a
DNA probe, an RNA probe, or a protein probe. The method may also
include analyzing the output current from the sensing nanoelectrode
to quantify a number of methylation sites in the oligonucleotide.
The method may also include applying a rate control bias to a rate
control nanoelectrode in the 3D nanopore device to modulate a
translocation rate of the oligonucleotide through the nanochannel.
The current may be an electrode current or a tunneling current.
[0022] In one or more embodiments, the first gating nanoelectrode
addresses a first end of the nanochannel, the second gating
nanoelectrode addresses a second end of the nanochannel opposite
the first end, and a sensing nanoelectrode addresses a first
location in the nanochannel between the first and second ends. The
method may also include alternatively reversing the electrophoretic
bias and the selection bias to direct alternating flow of the
oligonucleotides through the nanochannel between the first and
second gating nanoelectrodes.
[0023] In one or more embodiments, the 3D nanopore device is
integrated into a mobile application, a laptop computer, or a
desktop computer. The 3D nanopore device may be integrated into
microfluidic device, a nanofluidic device, a nanodevice, or a
lab-on-chip system. The 3D nanopore device may be integrated into
an all-in-one ASIC platform system for extraction and sensing of
the oligonucleotide.
[0024] In one or more embodiments, the method also includes the 3D
nanopore device detecting hybridization of the oligonucleotide to
the oligonucleotide probe at a minimum concentration of the
oligonucleotide of about 10 femtomolar (limit of detection). The
method may also include the 3D nanopore device detecting
hybridization of the oligonucleotide to the oligonucleotide probe
without amplification of the oligonucleotide or use of PCR. The 3D
nanopore device may be integrated into a liquid biopsy panel
platform to perform detection without amplification of the
oligonucleotide or use of PCR.
[0025] In one or more embodiments, the method also includes
analyzing the output current from the sensing nanoelectrode to
determine a conformation change of the oligonucleotide. The method
may also include analyzing the output current from the sensing
nanoelectrode to determine a hydration change of the
oligonucleotide.
[0026] In another embodiment, a method of determining an
oligonucleotide conformation change includes providing a 3D
nanopore device having top and bottom chambers, and a 3D
nanochannel array disposed in the top and bottom chambers such that
the top and bottom chambers are fluidly coupled by a plurality of
nanochannels in the 3D nanochannel array. The method also includes
purifying an oligonucleotide. The method further includes
functionalizing the 3D nanochannel array by coupling an
oligonucleotide probe to an inner surface of the 3D nanopore device
defining the nanochannel, where the oligonucleotide probe is
complementary to the oligonucleotide. Moreover, the method includes
adding the purified oligonucleotide to DI water to form an
oligonucleotide solution having a known concentration. In addition,
the method includes adding the oligonucleotide solution including
the oligonucleotide to the top and bottom chambers. The method also
includes placing top and bottom electrodes in the top and bottom
chambers respectively. The method further includes applying an
electrophoretic bias between the top and bottom electrodes.
Moreover, the method includes applying a selection bias across
first and second gating nanoelectrodes in the 3D nanopore device to
direct flow of the oligonucleotide through a nanochannel of the
plurality of nanochannels. In addition, the method includes
applying a sensing bias through a sensing nanoelectrode in the 3D
nanopore device. The method also includes detecting an output
current from the sensing nanoelectrode. The method further includes
analyzing the output current from the sensing nanoelectrode to
determine a conformation change of the oligonucleotide.
[0027] In still another embodiment, a method of determining an
oligonucleotide hydration change includes providing a 3D nanopore
device having top and bottom chambers, and a 3D nanochannel array
disposed in the top and bottom chambers such that the top and
bottom chambers are fluidly coupled by a plurality of nanochannels
in the 3D nanochannel array. The method also includes purifying an
oligonucleotide. The method further includes functionalizing the 3D
nanochannel array by coupling an oligonucleotide probe to an inner
surface of the 3D nanopore device defining the nanochannel, where
the oligonucleotide probe is complementary to the oligonucleotide.
Moreover, the method includes adding the purified oligonucleotide
to DI water to form an oligonucleotide solution having a known
concentration. In addition, the method includes adding the
oligonucleotide solution including the oligonucleotide to the top
and bottom chambers. The method also includes placing top and
bottom electrodes in the top and bottom chambers respectively. The
method further includes applying an electrophoretic bias between
the top and bottom electrodes. Moreover, the method includes
applying a selection bias across first and second gating
nanoelectrodes in the 3D nanopore device to direct flow of the
oligonucleotide through a nanochannel of the plurality of
nanochannels. In addition, the method includes applying a sensing
bias through a sensing nanoelectrode in the 3D nanopore device. The
method also includes detecting an output current from the sensing
nanoelectrode. The method further includes analyzing the output
current from the sensing nanoelectrode to determine a hydration
change of the oligonucleotide.
[0028] The aforementioned and other embodiments of the invention
are described in the Detailed Description which follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] The drawings described below are for illustration purposes
only. The drawings are not intended to limit the scope of the
present disclosure. The drawings illustrate the design and utility
of various embodiments of the present disclosure. It should be
noted that the figures are not drawn to scale and that elements of
similar structures or functions are represented by like reference
numerals throughout the figures. In order to better appreciate how
to obtain the recited and other advantages and objects of various
embodiments of the disclosure, a more detailed description of the
present disclosure will be rendered by reference to specific
embodiments thereof, which are illustrated in the accompanying
drawings. Understanding that these drawings depict only typical
embodiments of the disclosure and are not therefore to be
considered limiting of its scope, the disclosure will be described
and explained with additional specificity and detail through the
use of the accompanying drawings.
[0030] FIG. 1 schematically illustrates a prior art solid-state 2D
nanopore device;
[0031] FIGS. 2 to 4 schematically illustrate 3D nanopore devices
according to various embodiments.
[0032] FIGS. 5 to 11 schematically depict a method for detecting
DNA methylation using a 3D nanopore device according to some
embodiments.
[0033] FIGS. 12A and 12B schematically depict a method for
manufacture a nanopore device according to some embodiments.
[0034] FIG. 13 is a 3D histogram illustrating a relationship
between a percentage of DNA methylation and an output current in a
nanopore methylation detection device according to some
embodiments.
[0035] FIG. 14 is a flow-chart depicting a method of detecting
methylation of oligonucleotides using a nanopore detection system
according to some embodiments.
[0036] FIG. 15 schematically depicts a mechanism of
detecting/classifying methylation of DNA in a 3D nanopore
device/sensor according to some embodiments.
[0037] FIGS. 16-18 schematically illustrate conformational changes
of double stranded DNA inside a 3D nanopore device/sensor according
to some embodiments.
[0038] FIG. 19 schematically illustrates a hydration mediated
mechanism of signal change in double stranded DNA inside a 3D
nanopore device/sensor according to some embodiments.
[0039] In order to better appreciate how to obtain the
above-recited and other advantages and objects of various
embodiments, a more detailed description of embodiments is provided
with reference to the accompanying drawings. It should be noted
that the drawings are not drawn to scale and that elements of
similar structures or functions are represented by like reference
numerals throughout. It will be understood that these drawings
depict only certain illustrated embodiments and are not therefore
to be considered limiting of scope of embodiments.
DETAILED DESCRIPTION OF ILLUSTRATED EMBODIMENTS
[0040] Methods are described herein to achieve amplification-free
and rapid detection of DNA methylation (e.g., in less than 10
minutes). Nanopore electrically assisted DNA methylation detection
devices that efficiently and effectively detect DNA methylation by
manipulating potentials to increase hybridization of DNA and
detecting electrical characteristics generated by hybridization of
methylated DNA are described herein. Such detection devices and
methods can be used in various biomolecular arrays, including
microarrays, CMOS arrays, and nanopore arrays (e.g., solid-state,
and hybrid nanopore arrays). Such detection devices and methods can
also be used with various multi-channel nanopore arrays, including
the 3D multi-channel nanopore arrays described above and planar
multi-channel nanopore arrays.
[0041] Multi-channel nanopore arrays that allow parallel processing
of DNA methylation detection may be used to achieve
amplification-free and rapid methylation detection. Examples of
such multi-channel nanopore arrays are described in U.S.
Provisional Patent Application Ser. Nos. 62/566,313 and 62/593,840,
and U.S. Utility patent application Ser. No. 16/524,033, the
contents of which have been previously incorporated by reference.
Such multi-channel nanopore arrays can be electrically addressed to
direct charged particles (e.g., methylated DNA) to specific
channels in these multi-channel nanopore arrays. Other arrays are
coupled to microfluidic channels outside the array. Electrically
addressing and sensing individual nanopore channels within
multi-channel nanopore arrays, as described in U.S. Provisional
Patent Application Ser. No. 62/612,534, the contents of which have
been previously incorporated by reference, can facilitate more
efficient and effective use of multi-channel nanopore arrays to
achieve low cost, high throughput, amplification-free, and rapid
detection of methylated DNA.
Mechanism of Characterization
[0042] In some embodiments, the mechanism of characterization
(e.g., sensing mechanism) of methylation patterns leverages certain
properties of oligonucleotide bases (e.g., in DNA molecules). The
guanine base is one of the four base pairs in the DNA molecules,
which is easily oxidized. In terms of energy levels, the electrical
charge of a guanine base is just 0.2 eV. Therefore the electrical
charge of the guanine base can migrate easily along a DNA chain
into the next oxidizing group or the next guanosine. The electrical
charges/energies of guanine-cytosine ("G-C") and adenine-thymine
("A-T") base pairs in DNA function as relative charge carriers,
allowing an electrical charge (e.g., of a guanine base) to hop
along the length of the DNA molecule between charge carriers. A
positively charged "hole" in a DNA molecule may have a lower energy
at one or more G-C sites and this hole may move from one G-C pair
to the next by coherent tunneling through the A-T sites in the DNA
molecule. As such, one or more positively charged holes in a DNA
molecule can affect (e.g., reduce the negative charge of) the
charge of the entire DNA molecule.
[0043] The mechanism of characterization (e.g., sensing mechanism)
of methylation patterns may also leverage hydration effects on
electrical fields of DNA molecules. In some embodiments, the
mechanism of characterization (e.g., sensing mechanism) is
performing in a de-ionized ("DI") water solution of
oligonucleotides (e.g., a DNA strand) such that water molecules and
oligonucleotides form hydrated bio-interfaces that effect
electrical charge characteristics. The hydrophobicity of DNA base
pairs and the DNA double helix results in a structure that
positions the hydrophobic DNA base pairs away from the water in a
DI water solution. The negatively charged backbone of the DNA
strand attracts positively charged ions around the backbone.
Methylation adds a methyl group (e.g., to cytosine), resulting in
an almost neutral energy level that can cover the negative charge
of the DNA backbone. Further, water molecules in a DI water
solution can form a water shell around the DNA strand in the
hydration state.
[0044] The mechanism of characterization (e.g., sensing mechanism)
of methylation patterns may also leverage charge effects of
hydration of DNA molecules in CpG islands. When hydrogen atoms
(e.g., from water molecules) face the phosphate backbone of the
DNA, they affect each other. The neutral nature of methyl groups
added during methylation, and their interface with the water
molecules result in a DNA backbone covered by hydrogen atoms.
Accordingly, these methylation mediated interactions can be
detected by their effect on the charge of the DNA molecule, which
can be sensed by imbedded electrodes. The 3-dimensional ("3D")
sensors described herein and methods using same are capable of
sensing the charge changes in the reaction chamber, which include
the total charge of the DNA molecules in solution.
[0045] Methylation of DNA (e.g., cytosine in C-G pairs) also
affects the stiffness of the methylated dinucleotides (e.g.,
deformational mode dependent effects). Methylation increases the
stiffness of the dinucleotides marginally, but increases the
stiffness of the neighboring dinucleotides more significantly.
Stiffening is further enhanced for consecutively methylated
dinucleotides, which may result in the effect of hypermethylation.
Steric interactions between the added methyl groups and the
nonpolar groups of the neighboring nucleotides may be responsible
for the stiffening in many embodiments. Hydration maps show that
methylation also alters the surface hydration structure in various
ways. Resistance to deformation of methylated DNA may contribute to
the stiffening of DNA for deformational modes lacking steric
interactions. The effect of methylation on the conformational
behavior of DNA may depend on the local sequence around the
methylation site.
[0046] Some embodiments of mechanisms of characterization (e.g.,
sensing mechanism) of methylation patterns described herein are
based at least partially on DNA hydration and the methyl group
neutralization of the DNA backbone, which may affect the H+ and OH-
groups in the reaction chamber. Some 3D nanopore sensor arrays
described herein facilitate detection of methylation with increased
sensitivity and reduced detection limit by decreasing the Debby
lenses of the sensing areas in the nanochannels.
[0047] One exemplary approach for measuring or sensing the DNA is
to analyze fluctuations in helicoidal parameters as indicated by
electrical signals measured by imbedded electrodes inside a 3D
nanopore sensor arrays, as described herein, DNA conformation
change is one of the mechanisms that can alter the charge in the
electrode area and generate a signal. DNA methylation results in
weak fluctuations in the DNA structure resulting in stiffer DNA.
Also, methylation adds methyl groups that change the hydration
environment of the DNA molecule adjacent the methylation site.
These various mechanisms reduce the solving energy for better
characterization of hydration shells around the methyl group.
[0048] Because water has a high affinity for hydroxymethylcyosine
("hmC"), G-hmC base pairs experience the largest charge
fluctuations. In contrast, water is less apt to solvate the
hydrophobic methyl group of methylcytosine ("mC"), which increases
the rigidity/inflexibility of G-mC base pairs. Methylation of
cytosines in the substitute sequences
poly(deoxyguanylic-deoxycytidylic) acid sodium salt ("poly(dG-dC)")
permits the development of Z-DNA at more vulnerable ionic quality
than is required for unmethylated DNA.
[0049] In other embodiments, the output current mean values vary
according to the pattern: hmC<C<mC. Consequently, there may
be differences in DNA adaptability to methylation.
Exemplary Nanopore Devices
[0050] FIG. 2 schematically depicts a nanopore device 200 with a
three dimensional ("3D") array architecture according to one
embodiment. The device 200 includes a plurality of 2D arrays or
layers 202A-202D stacked along a Z axis 204. While the 2D arrays
202A-202D are referred to as "two dimensional," each of the 2D
arrays 202A-202D has some thickness along the Z axis.
[0051] The top 2D array 202A includes first and second selecting
(inhibitory nanoelectrode) layers 206, 208 configured to direct
movement of charged particles (e.g., biopolymers) through the
nanopores 210 (pillars, nanochannels) formed in the first and
second selecting layers 206, 208. The first selecting layer 206 is
configured to select from a plurality of rows (R1-R3) in the 2D
array 202A. The second selecting layer 208 is configured to select
from a plurality of columns (C1-C3) in the 2D array 202A. In one
embodiment, the first and second selecting layers 206, 208 select
from the rows and columns, respectively, by modifying a charge
adjacent the selected row and column and/or adjacent to the
non-selected rows and columns. The other 2D arrays 202B-202D
include rate control/current sensing nanoelectrodes. Rate
control/sensing nanoelectrodes may be made of highly conductive
metals and polysilicon, such as Au--Cr, TiN, TaN, Ta, Pt, Cr,
Graphene, Al--Cu, etc. The rate control/sensing nanoelectrodes may
have a thickness of about 0.3 to about 1000 nm. Rate
control/sensing nanoelectrodes may also be made in the biological
layer in hybrid nanopores. Each sensing nanoelectrode may be
operatively coupled/address to a nanopore 210 pillar, such that
each nanopore 210 pillar may be operatively coupled to a particular
memory cell. Electrical addressing in nanopore devices is described
in U.S. Provisional Patent Application Ser. No. 62/612,534, the
contents of which have been previously incorporated by
reference.
[0052] Hybrid nanopores include a stable biological/biochemical
component with solid-state components to form a semi-synthetic
membrane porin to enhance stability of the nanopore. For instance,
the biological component may be an .alpha.HL molecule. The
.alpha.HL molecule may be inserted into a SiN based 3D nanopore.
The .alpha.HL molecule may be induced to take on a structure to
ensure alignment of the .alpha.HL molecule with the SiN based 3D
nanopore by apply a bias to a nanoelectrode (e.g., in the top 2D
array 202A).
[0053] The nanopore device 200 has a 3D vertical pillar stack array
structure that provides a much larger surface area for charge
detection than that of a conventional nanopore device having a
planar structure. As a charged particle (e.g., biopolymer) passes
through each 2D array 202A-202E in the device, its charge can be
detected with a detector (e.g., nanoelectrode) in some of the 2D
arrays 202B-202E. Therefore, the 3D array structure of the device
200 facilitates higher sensitivity, which can compensate for a low
signal detector/nanoelectrode. The integration of memory cells into
the 3D array structure minimizes any memory related performance
limitations (e.g., with external memory device). Further, the
highly integrated small form factor 3D structure provides a high
density nanopore array while minimizing manufacturing cost.
[0054] In use, the nanopore device 200 is disposed between and
separating top and bottom chambers (not shown) such that the top
and bottom chambers are fluidly coupled by the nanopore pillars
210. The top and bottom chambers include a nanoelectrode (e.g.,
Ag/AgCl2, etc.) and a buffer (electrolyte solutions or DI water
with KCl) containing the charged particles (e.g., DNA) to be
detected. Different nanoelectrodes and electrolyte solutions can be
used for the detection of different charged particles.
[0055] Electrophoretic charged particle translocation can be driven
by applying a bias to nanoelectrodes disposed in a top chamber (not
shown) adjacent the top 2D array 202A of the nanopore device 200
and a bottom chamber (not shown) adjacent the bottom 2D array 202E
of the nanopore device 200. In some embodiments, the nanopore
device 200 is disposed in a between top and bottom chambers (not
shown) such that the top and bottom chambers are fluidly and
electrically coupled by the nanopore pillars 210 in the nanopore
device 200. The top and bottom chambers may contain the electrolyte
solution.
[0056] FIG. 3 schematically depicts a nanopore device 300 according
to one embodiment. The nanopore device 300 includes an insulating
membrane layer (Si3N4) followed by row and column select
(inhibitory nanoelectrodes) 306 and 308, respectively (e.g., metal
or doped polysilicon), and a plurality (1st to Nth) of cell
nanoelectrodes 310 (e.g., metal or doped polysilicon). The
nanoelectrodes 306, 308, 310 of the nanopore device 300 are covered
by an insulator dielectric film 312 (e.g., Al.sub.2O.sub.3,
HfO.sub.2, SiO.sub.2, ZnO).
[0057] As shown in FIG. 4, when a translocation rate control bias
signal 410 for column and row voltages (e.g., Vd) is applied to the
3D nanopore sensor array 400, row and column inhibitory
voltage/bias pulses are followed by a verify (sensing) voltage/bias
pulse (e.g., Vg1, Vg2), as described herein. Vg3 and following
electrodes (Vg4.about.VgN) are sensing and translocation
electrodes. An exemplary signal 410 is depicted in FIG. 4 overlaid
on top of the 3D nanopore sensor array 400. Inhibitory biases are
applied to deselect various column and row nanopore pillar
channels/nanochannels, respectively. During sensing operation, both
column and row (inhibitory) select nanoelectrodes are selected. The
resulting surface charge 412 can be detected as a change in an
electrical characteristic, such as current.
[0058] In some embodiments, the nanoelectrodes can detect current
modulations using a variety of principles, including ion blockade,
tunneling, capacitive sensing, piezoelectric, and
microwave-sensing. It is also possible that ionic concentration or
so called ionic current change in the electrode (detected by the
reference electrode) can be amplified and accurately sensed by the
attached CMOS transistor as shown in the FIG. 4.
Exemplary Nanopore Electrically Assisted DNA Methylation Detection
Device and Method
[0059] FIG. 5 depicts a nanopore electrically assisted DNA
methylation detection device according to some embodiments. While a
portion of a nanopore detection device 500 including a single
nanochannel 510 is depicted in FIG. 5, nanopore electrically
assisted DNA methylation (e.g., epigenetic change) detection
devices can include a 3D array having a plurality of nanochannels.
DNA methylation sensing structure such as the nanopore detection
device 500 depicted in FIG. 5 leverage the charge sensitivity of
the nanochannels and the large surface area resulting from parallel
processing and 3D arrays to facilitate rapid amplification-free
detection of DNA methylation.
[0060] The nanopore detection device 500 includes nanoelectrodes
522, 524, 526, 528. These nanoelectrodes 522, 524, 526, 528 are
independently electrically addressed to control flow through the
nanochannel 510 (first and second gating nanoelectrodes 522, 524)
and detect charges in the nanochannel 510 (first and second sensing
nanoelectrodes 526, 528).
[0061] The nanopore detection device 500 also includes probes (PNA,
DNA morpholino oligomers) 532 that are coupled to an interior
surface 530 of the nanochannel 510. The interior surface 530 can
include Al.sub.2O.sub.3. The Al.sub.2O.sub.3 includes a large
number of hydroxyl groups to facilitate functionalization for
immobilization of probes 532 on the interior surface 530 of the
nanochannel 510. The probes 532 can be generated using known
molecular biology techniques to be complementary to the target
region within genomic DNA (e.g., CpG islands in a promoter region).
The probes (e.g., DNA, RNA, PNA, LNA, Morpholinos, etc.) 532 can
have a variety of lengths (e.g., 24 base pairs, 40 base pairs,
etc.)
[0062] The probes 532 can be coupled/covalently bonded to the
interior surface using vapor-phase silanization. The thickness of
the organic coating of probes 532 can also be modulated by
modifying the time of the vapor-phase silanization.
[0063] In some embodiments, the nanopore device is first treated
with O.sub.2 plasma to generate --OH groups on the oxide dielectric
(Al.sub.2O.sub.3, HfO.sub.2, etc.) Al.sub.2O.sub.3 substrate
thereby activating the substrate for attaching target functional
groups. Then, 3-aminopropyl triethoxy silane (APTES) is used for
silanization because it is effective on a variety of possible
surface structures and because it is extremely reactive. Before
covalent attachment of the probes 532, the nanopore device 510 is
exposed to silanes (e.g., APTES And OTMS 1:3 ratio in ethanol) in
vapor phase by placing it in a dynamically pumped low vacuum
chamber adjacent a glass holder containing 50 .mu.l of APTES (from
Sigma-Aldrich), at ambient temperature and a base pressure of about
30 kPa. Then, the nanopore device 510 is removed from the vacuum
chamber and immersed in a 2.5% glutaraldehyde solution
(Sigma-Aldrich) for one hour. Next the nanopore device 510 is
removed from the cross-linker and washed twice in IPI and twice in
double distilled water. Finally, the nanopore device 510 is treated
(e.g., by immersion) overnight at 37.degree. C. with a 100 nM
amino-modified probe. After each step, the nanopore device is
washed in Ultrapure DNase/RNase-Free Distilled water (used as
washing buffer). Using such methods, covalent
attachment/immobilization of the probes 532 can be accomplished in
approximately 24 hours, or in eight hours at 45.degree. C.
[0064] The sensitivity of the nanopore detection device 500
hybridization of electrically target biomolecules 540 (e.g.,
methylated oligonucleotides) to the probes 532 covalently bonded to
the interior surface 530 of the nanochannel 510 is such that a
single base mismatch can be detected based on the resulting
difference in electrical charge. The parallel processing resulting
from the 3D array structure of nanopore devices dramatically
increases the interface area between the nanopore devices and the
methylated oligonucleotides to be detected, thereby increasing
sensitivity to a level sufficient for a point of care diagnosis and
determination of prognosis of a variety of disorders (e.g., genetic
disorders).
[0065] The first and second gating nanoelectrodes 522, 524 are
independently addressed and can therefore be rapidly electrically
modified to generate a "ping-pong" movement of target biomolecules
540 that increases hybridization of the target biomolecules 540 and
the probes 532. A potential across the first and second gating
nanoelectrodes 522, 524 in the nanochannel 510 can be rapidly
reversed by applying current to the first and second gating
nanoelectrodes 522, 524. The first and second gating nanoelectrodes
522, 524 can also be addressed to control translocation of target
biomolecules 540 through the nanochannel 510.
[0066] The target charge biomolecules 540 can be many varieties of
nucleic acids such as DNA, cDNA, mRNA, etc. The probes 532 can be
complementary DNA strands, locked nucleic acid (LNA) oligomers,
neutral backbone oligomers like peptide nucleic acids (PNA), DNA
morpholino oligomers, or any type of complementary strands that can
hybridize with the target charge biomolecules 540.
[0067] As shown in FIG. 5, before any current/potential is applied
to the nanopore detection device 500, the target biomolecules 540
are not attracted to the nanochannel 510. FIG. 6 depicts
application of current to generate a positive potential in the
first and second gate nanoelectrodes 522, 524. This positive
potential attracts the negatively target biomolecules 540 toward
the nanochannel 510.
[0068] FIG. 7 depicts continued application of current to generate
a positive potential in the first and second gate nanoelectrodes
522, 524. Over time, some of the negatively target biomolecules 540
enter the nanochannel 510, and interact with the probes 532
covalently bonded to the interior surface 530 of the nanochannel
510. This interaction between the negatively target biomolecules
540 and the probes 532 results in hybridization between the two
molecules. This electrically connects the negatively target
biomolecules 540 to the first and second sensing nanoelectrodes
526, 528, which can detect the negative charges 534 associated with
the negatively target biomolecules 540.
[0069] FIG. 5 depicts a modification of the electrical potentials
in the first and second gate nanoelectrodes 522, 524. In FIG. 5,
current is no longer applied to the first gate nanoelectrode 522,
eliminating the positive potential therein. However, current is
maintained across the second gate nanoelectrode 524 to maintain a
positive potential therein. This change in potential draws the
negatively target biomolecules 540 in the nanochannel 510 toward
the second gate nanoelectrode 524, as indicated by the flow arrow
550. FIG. 5 also shows that more negatively target biomolecules 540
have hybridized to the probes 532 in the nanochannel 510.
[0070] FIG. 9 depicts another modification of the electrical
potentials in the first and second gate nanoelectrodes 522, 524. In
FIG. 9, current is no longer applied to the second gate
nanoelectrode 524, eliminating the positive potential therein.
However, current is applied across the first gate nanoelectrode 522
to maintain a positive potential therein. This change in potential
draws the negatively target biomolecules 540 in the nanochannel 510
back toward the first gate nanoelectrode 522, as indicated by the
flow arrow 552. FIG. 9 also shows that, with more exposure of the
charge biomolecules 540 to the probes 532 in the nanochannel 510,
even more negatively target biomolecules 540 have hybridized to the
probes 532.
[0071] FIG. 10 depicts still another modification of the electrical
potentials in the first and second gate nanoelectrodes 522, 524. In
FIG. 9, current is no longer applied to the first gate
nanoelectrode 522, eliminating the positive potential therein.
However, current is applied across the second gate nanoelectrode
524 to maintain a positive potential therein. This change in
potential draws the negatively target biomolecules 540 in the
nanochannel 510 back toward the second gate nanoelectrode 524, as
indicated by the flow arrow 550. FIG. 10 also shows that, with even
more exposure of the charge biomolecules 540 to the probes 532 in
the nanochannel 510, still more negatively target biomolecules 540
have hybridized to the probes 532.
[0072] The direction changes depicted in the flow arrows 550, 552
in FIGS. 5 to 10 depict the first two direction changes in the
"ping-pong" movement of target biomolecules 540 that increases
hybridization of the target biomolecules 540 and the probes 532.
The direction changes are controlled by changing the electrical
potentials in the first and second gate nanoelectrodes 522, 524,
which is in turn modified by alternating the current applied
thereto. Because currents can be applied to the individually
electrically addressed first and second gate nanoelectrodes 522,
524 under processor control, the alternation of current and
electrical potentials can be executed rapidly. The "ping-pong"
movement of charged biomolecules 540 increases the amount of time
the charged biomolecules 540 are exposed to the probes 532 in the
nanochannel 510, thereby increasing the amount of hybridization
between the two molecules. While only one or two changes of
direction are depicted in FIGS. 5 to 10, a biomolecule detection
method can include many more changes of direction to increase the
hybridization of the target biomolecules 540.
[0073] FIG. 11 depicts the end of a series of "ping-pong" movements
in a biomolecule detection method. At the end of the detection
method, a plurality of negatively target biomolecules 540
(methylated oligonucleotide) have hybridized to the probes 532,
which are themselves covalently bonded to the interior surface 530
of the nanochannel 510. As each negatively target biomolecules 540
hybridizes to a probe 532, its additional negative charge 534 is
detected by the first and/or second sensing nanoelectrode 526, 528.
The sensing nanoelectrodes 526, 528 are sufficiently sensitive to
distinguish single base pair mismatches. Therefore, the sensing
nanoelectrodes 524, 528 can detect the negative charges 534
associated with hybridization of each target biomolecules 540. As
such, the nanopore detection device 500 can rapidly (e.g., under 10
minutes) detect and quantitate target DNA methylation in a
solution.
[0074] While the nanopore detection device 500 depicted in FIGS. 5
to 11 is configured to detect only a single negatively charged
target biomolecules 540 during a particular procedure, nanopore
detection devices according to other embodiments can be configured
to detect multiple negatively charged target biomolecules (e.g.,
methylated oligonucleotides). Such nanopore detection devices
include a plurality of probes that (1) hybridized with different
negatively charged target biomolecules and (2) have different
lengths. Because the probes have different lengths, hybridization
of different negatively charged target biomolecules will result in
a different amount of negative charge being electrically added to
the interior surface of the nanochannel. The sensing nanoelectrodes
are sufficiently sensitive to distinguish these different amounts
of negative charge, and thereby distinguish hybridization of
different negatively charged target biomolecules.
Exemplary Nanopore Device Manufacturing Method
[0075] FIGS. 12A and 12B schematically depict a method 1210 for
manufacture a nanopore device, such as the nanopore detection
devices 500, 600 described above, according to some
embodiments.
[0076] At step 1212, an interior surface of the nanopore device (in
the nanochannel) is O.sub.2 plasma treated, cleaned, and activated.
At step 1214, the surface of the device is silanized by treating
with (3-aminopropyl)triethoxysilane (APTES) to functionalize the
surface. At step 1216, an aldehyde linker is attached to the
functionalized surface. At step 1218 (FIG. 12B), a probe (e.g.,
PNA) is attached to the surface via the aldehyde. At step 1220, the
negatively charged target biomolecule (e.g., methylated DNA)
attaches to the probe on the surface and changes the charge of the
surface for electrically detecting the negatively charged target
biomolecule, as described above.
Methylation Effect on Output Current
[0077] FIG. 13 is a 3D histogram 1300 showing measured output
current 1312 vs. applied sensing bias 1310 for a variety of
methylation percentages 1314 (for an oligonucleotide complementary
to an oligonucleotide probe). Five types of control DNA samples
containing different percentage of the methylation 0%, 12.5%, 25%,
50% and 100% 1314 were prepared and complementary probes were
designed. After simple functionalization with APTES, a
glutaraldehyde linker was added, and the probes were incubated into
particular locations in the 3D nanopore sensor arrays. Real time
measurements of output currents 1312 for different concentrations
of DNA methylation 1314 were performed at a variety of sensing
biases 1310 and the results summarized in FIG. 13. As shown in FIG.
13, as the percentage of methylation 1314 increases, the
signal/output current decreases 1312 (e.g., due to neutralization
of the negative backbone of the DNA and water methyl
interaction).
Blocking Electron Transfer
[0078] FIG. 15 schematically depicts the mechanism of the
detecting/classifying methylation of DNA in a 3D nanopore
device/sensor 1500 according to some embodiments. 1501 represents a
gate electrode, 1502 represents a dielectric layer with silane.
1503 represents a bond between a designed oligonucleotide probe
strand 1505 and a surface of the 3D nanopore device/sensor 1500.
1504 represents an electron transfer between guanine bases. 1506
represents the different hydrogen bounding between A-T and G-C base
pairs. 1507 represents a target sequence from a clinical sample,
which carries methyl groups. The target sequence/oligonucleotide
strand 1507, which has been methylated to a certain degree, is
complementary to the oligonucleotide probe strand 1505, and
therefore bonds thereto; As shown at 1508, the electron pathway
from base to base is blocked by a methyl group 1509 (e.g., in
methyl cytosine). This blockage reduces the output current measured
by the gate electrode 1501 of the 3D nanopore device/sensor 1500.
The amount of reduction is related to the percentage of methylation
of the target sequence 1507 (as shown in FIG. 13).
[0079] The top embodiment in FIG. 15 illustrates that, when a
positive gate bias is applied to the gate electrode 1501 in the 3D
nanopore device/sensor 1500, electrons in the oligonucleotide probe
1505, which is attached to the surface of the device 1500, migrate
to the gate electrode 1501. The electrons migrate 1504 between the
most easily oxidized sites in the DNA strand 1505, which are
guanine bases. The electrons continue to migrate to the next easily
oxidized base through the DNA strand 1505, which is next guanine
base, until it reaches the gate electrode 1501, which the electrons
are sensed (e.g., as an output current).
[0080] The bottom embodiment in FIG. 15 illustrates that, when a
target sequence/oligonucleotide strand 1507 is added to the 3D
nanopore device/sensor 1500, the target oligonucleotide strand 1507
bonds to the oligonucleotide probe 1505. After attachment of the
target oligonucleotide strand 1507, when a positive gate bias is
applied to the gate electrode 1501, the methyl groups 1509 in the
methylated cytosine based interrupts the electron transfer
mechanism, reducing electron transfer and signal depending on the
percentage of methylation of the target oligonucleotide strand
1507. The measured electrical signal (e.g., output current) can be
compared with reference methylation percentage profiles (see FIG.
13) to determine the methylation pattern of target oligonucleotide
strand 1507.
Conformational Changes
[0081] In some embodiments, methylated and un-methylated
oligonucleotides have different conformations, with methylation
resulting in a conformation change. The different conformation of a
methylated oligonucleotide may change the charge signal at the
surface of a 3D nanopore device/sensor electrode. The change in
surface charge signal may result in changes in the signal read by
the electrode (e.g., output current). The measured changes in the
signal may be analyzed to determine conformational changes.
[0082] FIGS. 16-18 schematically illustrate conformational changes
of the double stranded DNA inside a 3D nanopore device/sensor 1600
according to some embodiments. 1601 represents an electrode (e.g.,
gate or sensing electrode) and surface structures of the device
1600, 1602 represents a dielectric layer with silane. 1602
represents a bonding site between a designed oligonucleotide probe
strand 1603 and a surface of the 3D nanopore device/sensor 1600.
1604 represents a target sequence/oligonucleotide strand. DNA
conformation/configuration can change based on the environment of
the DNA molecule. For instance, various ions can change the DNA
conformation/configuration into a different form of the
configuration. FIG. 16 shows the target sequence/oligonucleotide
strand 1604 in a B-DNA configuration. FIG. 17 shows the target
sequence/oligonucleotide strand 1604' in a Z-DNA configuration.
FIG. 18 shows the target sequence/oligonucleotide strand 1604'' in
a "hairpin" configuration. The 3D nanopore device/sensor 1600 can
measure signal changes when the target sequence/oligonucleotide
strand 1604, 1604', 1604'' bonds to an oligonucleotide probe 1603
in a DI water environment. These real time signal changes may be
analyzed to determine conformational changes.
Hydration Changes
[0083] In some embodiments, methylation may result in changes to
hydration of the oligonucleotide. Hydration changes may affect the
sensing mechanism by changing the oligonucleotide configuration
during hydrogen binding between the complimentary strands. The
configuration change may result in changes in the signal read by
the electrode (e.g., output current). The measured changes in the
signal may be analyzed to determine hydration changes.
[0084] FIG. 19 schematically illustrates the hydration mediated
mechanism of signal change in DNA molecules with methylated
cytosine bases according to some embodiments. Methylated cytosine
bases affect the extent of hydration of the target
sequence/oligonucleotide strand. The hydration changes, in turn,
affect the charge arrangements in the sequence/oligonucleotide
strand and the oligonucleotide probe. The 3D nanopore device/sensor
can measure signal changes when the target sequence/oligonucleotide
strand bonds to an oligonucleotide probe in a DI water environment.
These real time signal changes may be analyzed to determine
hydration changes.
Method of Detecting Methylation of DNA Using Nanopore Detection
System
[0085] With reference data such as that depicted in FIG. 13, the
nanopore detection systems described herein can be used in a method
of detecting methylation of oligonucleotides. For instance, FIG. 14
depicts a method 1400 of detecting methylation of oligonucleotides
using a nanopore detection system according to some embodiments. At
step 1410, a target oligonucleotide is purified. The target
oligonucleotide may be a CpG island in a promoter of a gene (e.g.,
a cancer suppressing gene).
[0086] At step 1412, a nanochannel is functionalized. The
nanochannel is in a 3D nanopore device having top and bottom
chambers, with the a 3D nanochannel array disposed in the top and
bottom chambers such that the top and bottom chambers are fluidly
coupled by a plurality of nanochannels in the 3D nanochannel array.
The nanochannel may be functionalized by coupling an
oligonucleotide probe to an inner surface of the 3D nanopore device
defining the nanochannel, wherein the oligonucleotide probe is
complementary to the oligonucleotide.
[0087] At step 1414, a DI water solution with the oligonucleotide
is added to the 3D nanopore device.
[0088] At step 1416, an electrophoretic bias is applied to top and
bottom electrodes in the top and bottom chambers of the 3D nanopore
device to drive charged particles through the nanochannels.
[0089] At step 1418, a selection bias is applied to first and
second gating nanoelectrodes in the 3D nanopore device to direct
flow of the oligonucleotide through a nanochannel of a plurality of
nanochannels in the 3D nanopore device.
[0090] At step 1420, a sensing bias is applied to a sensing
electrode in the 3D nanopore device to elicit an output
current.
[0091] At step 1422, an output current is detected from the sensing
electrode.
[0092] At step 1424, the output current from the sensing
nanoelectrode to determine a methylation percentage of the
oligonucleotide. For instance, the output current can be compared
to reference data such as that depicted in FIG. 13. Taking multiple
output current measurements while changing/sweeping the sensing
bias applied to the 3D nanopore device can improve the accuracy of
methylation percentage determination.
[0093] The nanopore detection systems described herein are 3D
sensors that work with DI water as a buffer. The function and exact
mechanism of action for water molecules within nanoscale small
spaces have not been previous investigated and understood, but
highly sensitive and clear resolution of the 3D arrays described
herein may prove the benefit of using DI water instead of
electrolytes or other buffer solutions, which increases the noise
level within such sensitive sensors.
[0094] The mechanism of reaction and signal generation in the
nanopore detection systems described herein is based on changing
the charge distribution in the surface because of hydration of
methylated DNA molecules that attach to the probes described above.
This hydration causes changes in the electrode with the
redistribution of charge density at the gate nanoelectrodes.
Nanoelectrodes inside of the nanopores have an all-around or
belt-like morphology surrounding the nanopore, which increases the
sensitivity of the nanopore sensor.
[0095] By using different potential gradients at each nanopore, a
user can control the speed of charged biomolecules traveling inside
and through each nanopore. Using a low concentration
buffer/electrolyte or DI water to increase the Debye length of the
sensing area in the nanopore is one of the unique properties of the
3D nanopore detection systems described herein. A user has broad
control over the nanopore detection system by changing the amount
and duration of electrical potential for each nanoelectrode to
electrophoretically control movement of the charged target
biopolymers and the Ping-Ponging motion of same between the
nanoelectrodes as described above. As described above, when charged
target biopolymers moves back and forth between nanoelectrodes with
changing/alternating nanoelectrode potential, time required for the
charged target biopolymers to attach to the probes will be reduces
to less than 10 minutes. This reduction in attachment time is due
to increased interaction between the targets and the probes,
allowing them to bond with each other in less time.
[0096] In some embodiments of nanopore detection systems, such as
those described herein, the size, shape, and depth of the nanopore
structure can be modified based on the size of the probe. For
instance, a pore size with a diameter of 50 nm (500 .ANG.) may be
used for sensing target biopolymers with a 40 bp probe. In other
embodiments, a pore size with a diameter of 100 nm may use for
sensing target biopolymers with more than 100 bp probes. In still
other embodiments, a pore size with a diameter of 200 nm may be
used for sensing target biopolymers with still longer probes.
[0097] The 3D nanopore array sensors described herein are more
sensitive and compact compared to 2D or planar structure sensors
because the 3D array of nanopores increases the surface to volume
ratio, allowing for miniaturization of the smart surfaces inside
the nanochannels of the nanopore arrays. The high surface to volume
ratio allows sensing of very low concentrations (e.g., 10
femtomolar) of DNA methylation.
[0098] The 3D nanopore array sensors described herein provide
better control compared to charge perturbation or electrochemical
based sensor systems because the dielectric layer insolates the
inner surfaces of each nanochannel, thereby enhancing the
capacitance effect and control of the electrical field effect for
each nanochannel.
[0099] The 3D nanopore array sensors described herein can use
capacitance variation for sensing DNA methylation with an
immobilized probe. When a target DNA molecule passes within a
nanopore of the array structure (electrophoretically driven by the
external voltage), the top and bottom electrodes record a change in
the potential resulting from the passing DNA molecule within the
nanopore structure, polarizing the nanopore like a capacitor. The
resulting capacitance variation can be measured electronically to
detect passage of the target DNA molecule. The speed of the DNA
molecule can be controlled by controlling the applied positive gate
biases, allowed the 3D nanopore array sensor to be used in
methylation detection. The 3D nanopore array sensors described
herein can detect passage of DNA methylation by detecting both
tunneling current and capacitance change. Previously existing
biological nanopores cannot detect tunneling current and
capacitance change because they do not have embedded nanoelectrodes
in their structure.
[0100] The probes used in the 3D nanopore array sensors described
herein may be modified to alter their surface chemistry, allowing
more system control and design options. For instance, thiol
modification may be used for thiol gold binding. Avidin/biotin and
EDC crosslinker/N-hydroxysuccinimide (NHS) are other probe
modification and target pairs that may be used with the 3D nanopore
array sensors described herein with modification of structure and
chemistry of immobilizing techniques.
[0101] The corresponding structures, materials, acts and
equivalents of all means or step plus function elements in the
claims below are intended to include any structures, materials,
acts and equivalents for performing the function in combination
with other claimed elements as specifically claimed. It is to be
understood that while the invention has been described in
conjunction with the above embodiments, the foregoing description
and claims are not to limit the scope of the invention. Other
aspects, advantages and modifications within the scope to the
invention will be apparent to those skilled in the art to which the
invention pertains.
[0102] Various exemplary embodiments of the invention are described
herein. Reference is made to these examples in a non-limiting
sense. They are provided to illustrate more broadly applicable
aspects of the invention. Various changes may be made to the
invention described and equivalents may be substituted without
departing from the true spirit and scope of the invention. In
addition, many modifications may be made to adapt a particular
situation, material, composition of matter, process, process act(s)
or step(s) to the objective(s), spirit or scope of the present
invention. Further, as will be appreciated by those with skill in
the art that each of the individual variations described and
illustrated herein has discrete components and features which may
be readily separated from or combined with the features of any of
the other several embodiments without departing from the scope or
spirit of the present inventions. All such modifications are
intended to be within the scope of claims associated with this
disclosure.
[0103] Any of the devices described for carrying out the subject
diagnostic or interventional procedures may be provided in packaged
combination for use in executing such interventions. These supply
"kits" may further include instructions for use and be packaged in
sterile trays or containers as commonly employed for such
purposes.
[0104] The invention includes methods that may be performed using
the subject devices. The methods may comprise the act of providing
such a suitable device. Such provision may be performed by the end
user. In other words, the "providing" act merely requires the end
user obtain, access, approach, position, set-up, activate, power-up
or otherwise act to provide the requisite device in the subject
method. Methods recited herein may be carried out in any order of
the recited events which is logically possible, as well as in the
recited order of events.
[0105] Exemplary aspects of the invention, together with details
regarding material selection and manufacture have been set forth
above. Other details of the present invention, these may be
appreciated in connection with the above-referenced patents and
publications as well as generally known or appreciated by those
with skill in the art. The same may hold true with respect to
method-based aspects of the invention in terms of additional acts
as commonly or logically employed.
[0106] In addition, though the invention has been described in
reference to several examples optionally incorporating various
features, the invention is not to be limited to that which is
described or indicated as contemplated with respect to each
variation of the invention. Various changes may be made to the
invention described and equivalents (whether recited herein or not
included for the sake of some brevity) may be substituted without
departing from the true spirit and scope of the invention. In
addition, where a range of values is provided, it is understood
that every intervening value, between the upper and lower limit of
that range and any other stated or intervening value in that stated
range, is encompassed within the invention.
[0107] Also, it is contemplated that any optional feature of the
inventive variations described may be set forth and claimed
independently, or in combination with any one or more of the
features described herein. Reference to a singular item, includes
the possibility that there are plural of the same items present.
More specifically, as used herein and in claims associated hereto,
the singular forms "a," "an," "said," and "the" include plural
referents unless the specifically stated otherwise. In other words,
use of the articles allow for "at least one" of the subject item in
the description above as well as claims associated with this
disclosure. It is further noted that such claims may be drafted to
exclude any optional element. As such, this statement is intended
to serve as antecedent basis for use of such exclusive terminology
as "solely," "only" and the like in connection with the recitation
of claim elements, or use of a "negative" limitation.
[0108] Without the use of such exclusive terminology, the term
"comprising" in claims associated with this disclosure shall allow
for the inclusion of any additional element--irrespective of
whether a given number of elements are enumerated in such claims,
or the addition of a feature could be regarded as transforming the
nature of an element set forth in such claims. Except as
specifically defined herein, all technical and scientific terms
used herein are to be given as broad a commonly understood meaning
as possible while maintaining claim validity.
[0109] The breadth of the present invention is not to be limited to
the examples provided and/or the subject specification, but rather
only by the scope of claim language associated with this
disclosure.
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